RESUMO
Inflammatory cytokines (IC) activate endothelial cell adhesiveness for monocytes and inhibit endothelial cell growth. Here we report the identification of the human guanylate binding protein-1 (GBP-1) as the key and specific mediator of the anti-proliferative effect of IC on endothelial cells. GBP-1 expression was induced by IC, downregulated by angiogenic growth factors, and inversely related to cell proliferation both in vitro in microvascular and macrovascular endothelial cells and in vivo in vessel endothelial cells of Kaposi's sarcoma. Experimental modulation of GBP-1 expression demonstrated that GBP-1 mediates selectively the anti-proliferative effect of IC, without affecting endothelial cell adhesiveness for monocytes. GBP-1 anti-proliferative activity did not affect ERK-1/2 activation, occurred in the absence of apoptosis, was found to be independent of the GTPase activity and isoprenylation of the molecule, but was specifically mediated by the C-terminal helical domain of the protein. These results define GBP-1 as an important tool for dissection of the complex activity of IC on endothelial cells, and detection and specific modulation of the IC-activated non-proliferating phenotype of endothelial cells in vascular diseases.
Assuntos
Proteínas de Ligação a DNA/química , Endotélio Vascular/efeitos dos fármacos , Proteínas de Ligação ao GTP/química , Mediadores da Inflamação/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Células Cultivadas/efeitos dos fármacos , DNA Antissenso/genética , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Endotélio Vascular/citologia , Proteínas de Ligação ao GTP/biossíntese , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/fisiologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Infecções por HIV/complicações , Humanos , Mediadores da Inflamação/farmacologia , Interferon gama/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Prenilação de Proteína , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , RNA Mensageiro/biossíntese , Proteínas Recombinantes de Fusão/fisiologia , Sarcoma de Kaposi/irrigação sanguínea , Sarcoma de Kaposi/etiologia , Sarcoma de Kaposi/patologia , Neoplasias Cutâneas/irrigação sanguínea , Neoplasias Cutâneas/etiologia , Neoplasias Cutâneas/patologia , Relação Estrutura-Atividade , Células U937/metabolismo , Veias UmbilicaisRESUMO
Patients with Kaposi's sarcoma (KS) have a human herpesvirus-8 (HHV-8) load higher than patients without KS and present a CD8(+) T-cell activation with production of Th1-type cytokines both in tissues and peripheral blood mononuclear cells (PBMC). Because in tissues of KS patients detection of inflammatory cytokines (IC) can precede detection of HHV-8 DNA and because signs of immunoactivation and/or dysregulation can precede KS development, we investigated the effect of IC on HHV-8 infection. To achieve this goal, PBMC and purified cell populations from 45 patients with KS and 45 patients at risk of KS were analyzed for HHV-8 DNA and/or gene expression and for cell survival, growth, and phenotype before or after culture with or without the IC increased in KS. The results indicate that PBMC that are polymerase chain reaction (PCR)-positive at day 0 generally loose the virus upon culture. However, the presence of IC maintains HHV-8 DNA load in cultured cells. In addition, IC increase viral load to detectable levels in PBMC from serologically positive patients that were PCR-negative before culture. gamma Interferon is sufficient for these effects, whereas tumor necrosis factor and interleukin-6 have little or no activity. The increase of HHV-8 DNA by IC is observed after short-term (7 days) or long-term (28 days) culture of the cells and occurs in one or both of the two circulating cell types that are infected in vivo: B cells and monocytes. In both cases it is associated with lytic gene expression, suggesting that virus reactivation is one of the most likely mechanisms for the effect of IC on virus load. However, IC have also effects on the cells target of HHV-8 infection, because they increase B-cell survival and induce the growth and differentiation of monocytes into KS-like spindle cells with markers of endothelial macrophages. Because cells with markers of endothelial macrophages are present in blood and lesions from KS patients and are infected by HHV-8, these data may explain the high HHV-8 load associated with KS development and suggest that infected monocytes may carry the virus to tissues, transmit the infection, or differentiate in loco in spindle cells with endothelial macrophage markers.
Assuntos
Linfócitos B/virologia , Citocinas/farmacologia , Herpesvirus Humano 8/crescimento & desenvolvimento , Monócitos/virologia , Sarcoma de Kaposi/metabolismo , Ativação Viral , Síndrome da Imunodeficiência Adquirida/complicações , Linfócitos B/citologia , Divisão Celular , Sobrevivência Celular , Células Cultivadas , Citocinas/metabolismo , DNA Viral/análise , Herpesvirus Humano 8/genética , Humanos , Masculino , Monócitos/citologia , Fenótipo , Reação em Cadeia da Polimerase , Sarcoma de Kaposi/complicações , Sarcoma de Kaposi/virologiaRESUMO
Analysis by polymerase chain reaction (PCR) and serological studies have demonstrated a close association between the novel human herpes virus, Kaposi's sarcoma-associated herpes virus (KSHV) or human herpes virus-8 (HHV-8) and the development of Kaposi's sarcoma (KS). To clarify the role of HHV-8 in KS pathogenesis, we investigated at the cellular level by in situ hybridization the expression of a recently described 0.7-kb HHV-8-encoded mRNA (T0.7 mRNA) in KS tissues of different epidemiological origin (AIDS-KS, African endemic KS and classical KS). The T0.7 mRNA likely encodes a small membrane protein, supposedly expressed in latently HHV-8-infected cells. Indeed, we detected T0.7 mRNA in virtually all cells of the cell line BCBL-1 established from a body cavity-based lymphoma (BCBL) and latently infected with HHV-8. In all KS biopsies examined, independent of their epidemiological type, the late-stage (nodular) KS tissues showed a high level of T0.7 mRNA expression in typical KS spindle cells but also in endothelial cells lining blood vessels, indicating latent HHV-8 infection of these cells. The presence of T0.7-expressing cells was restricted to KS tumor tissue and therefore appears to indicate an important role of latent HHV-8 infection in KS pathogenesis.
Assuntos
Síndrome da Imunodeficiência Adquirida/complicações , Herpesvirus Humano 8/isolamento & purificação , RNA Viral/isolamento & purificação , Sarcoma de Kaposi/virologia , África/epidemiologia , Biópsia , Epitélio/virologia , Herpesvirus Humano 8/genética , Humanos , Hibridização In Situ , Reação em Cadeia da Polimerase , Latência ViralRESUMO
Abundant vasculature with increased permeability is a prominent histological feature of Kaposi's sarcoma (KS), a multifocal, cytokine-regulated tumor. Here we report on the role of vascular endothelial growth factor (VEGF) in AIDS-KS angiogenesis and vascular permeability. We demonstrate that different cytokines, which were previously shown to be active in KS development, modulate VEGF expression in KS spindle cells and cooperate with VEGF on the functional level. Northern blot analysis as well as studies on single cells using in situ hybridization revealed that VEGF expression in cultivated AIDS-KS spindle cells is up-regulated by platelet-derived growth factor-B and interleukin-1 beta. Western blot and enzyme-linked immunosorbent assay analysis of cell culture supernatants demonstrated that the VEGF protein is secreted by stimulated AIDS-KS spindle cells in sufficiently high amounts to activate proliferation of human dermal microvascular endothelial cells. Basic fibroblast growth factor did not increase VEGF expression but acted synergistically with VEGF in the induction of angiogenic KS-like lesions in a mouse model in vivo. Angiogenesis and cellularity of KS-like lesions were clearly increased when both factors were injected simultaneously into the flanks of mice, compared with separate injection of each factor. A comparable angiogenic reaction as obtained by simultaneous injection of basic fibroblast growth factor and VEGF was observed when cell culture supernatants of AIDS-KS spindle cells were used for these experiments. Finally, analysis of primary human AIDS-KS lesions revealed that high amounts of VEGF mRNA and protein were present in KS spindle cells in vivo. These data provide evidence that VEGF, in concert with platelet-derived growth factor-B, interleukin-1 beta, and basic fibroblast growth factor, is a key mediator of angiogenesis and vascular permeability in KS lesions in vivo.